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. 2013 Feb 15;431(3):566-71.
doi: 10.1016/j.bbrc.2013.01.015. Epub 2013 Jan 11.

Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury

Lijuan Chen  1 , Yingjie Wang, Yaohua Pan, Lan Zhang, Chengxing Shen, Gangjian Qin, Muhammad Ashraf, Neal Weintraub, Genshan Ma, Yaoliang Tang
Affiliations

Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury

Lijuan Chen et al. Biochem Biophys Res Commun. .

Abstract

Background: Cardiac progenitors (CPC) mediate cardioprotection via paracrine effects. To date, most of studies focused on secreted paracrine proteins. Here we investigated the CPC-derived-exosomes on protecting myocardium from acute ischemia/reperfusion (MI/R) injury.

Methods and results: CPC were isolated from mouse heart using two-step protocol. Exosomes were purified from conditional medium, and confirmed by electron micrograph and Western blot using CD63 as a marker. qRT-PCR shows that CPC-exosomes have high level expression of GATA4-responsive-miR-451. Exosomes were ex vivo labeled with PKH26, We observed exosomes can be uptaken by H9C2 cardiomyoblasts with high efficiency after 12 h incubation. CPC-exosomes protect H9C2 from oxidative stress by inhibiting caspase 3/7 activation invitro. In vivo delivery of CPC-exosomes in an acute mouse myocardial ischemia/reperfusion model inhibited cardiomyocyte apoptosis by about 53% in comparison with PBS control (p<0.05).

Conclusion: Our results suggest, for the first time, the CPC-exosomes can be used as a therapeutic vehicle for cardioprotection, and highlights a new perspective for using non-cell exosomes for cardiac disease.

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Figures

Figure 1
Figure 1
Characterization of CPC and exosomes from CPC. (A) Mouse cardioprogenitor cells; (B) RT-PCR for mature cardiac marker (TnI) and early cardiac markers (Mesp1, GATA4 and Nkx2.5) in bone marrow derived mesenchymal stem cells (BM-MSC) and CPC. GATA4 mRNA was detected at high level by RT-PCR in CPC while absent in BM-MSC. (C) CPC-exosomes isolated by PEG precipitation; (D) electron micrograph image of CPC-derived exosomes. The image shows small vesicles of approximately 40-100 nm in diameter. Scale bar=100nm. (E) Western blot characterization of CPC-exosomes. CPC-exosome preparation was separated by SDS–PAGE, and electroblotted to the polyvinylidene fluoride (PVDF) membrane, and probed with exosome marker CD63.
Figure 2
Figure 2
uptake of exosomes by H9C2 and CPC-exosomes protecting H9C2 from H2O2 induced apoptosis in vitro (A) Uptake of PKH26-labeled-exosomes by H9C2 in vitro. H9C2 cells incorporated PKH26 labeled-exosomes from CPC after 12hrs incubation. Red dots indicate CPC-exosomes incorporated in H9C2 cells. (B) CPC-exosomes protected H9C2 from H2O2 induced apoptosis in vitro. Caspase-3/7 activity assay was used to assess the effect of protection of CPC-exosomes on H9C2 cells treated for 4hs of H2O2 (200μm). Data shown are mean ± SEM of 4 independent experiments.
Figure 3
Figure 3
Immunohistochemistry of control PBS or CPC-exosomes–treated heart sections marking TUNEL-positive cardiomyocytes costained with cardiac troponin I antibody within the border zone of infarcted hearts. (A and B) Representative illustration of TUNEL staining in heart section after 45min ischemia and 24h reperfusion. (C) Quantification of myocardial apoptosis. Green staining indicates TUNEL-positive cells , red staining indicating cTnI positive cardiomyocytes, Error bars indicate SEM (*, P < 0.05).
Figure 4
Figure 4
Real-time RT-PCR validation of differential expression of miR-451 and miR-144 in CPC-exosomes versus CPC. U6 snRNA was used as a normalization reference in real-time PCR
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